Crosstalk between cyclic-di-guanosine monophosphate and the sensor kinase MtrB regulates MtrA-dependent genes, bacterial growth, biofilm formation and lysosomal trafficking of Mycobacterium tuberculosis

Cyclic-di-guanosine monophosphate (c-di-GMP) plays an important role in bacterial signalling networks. C-di-GMP exerts a regulatory function through binding to diverse molecules that include transcription factors, riboswitches and sensor kinases (SKs), thereby regulating diverse processes. Here, we demonstrate the crosstalk between c-di-GMP and the SK MtrB of <i>Mycobacterium tuberculosis</i>. MtrB phosphorylates and regulates its cognate response regulator MtrA. C-di-GMP binds directly to the cytosolic domain of MtrB to inhibit its autophosphorylation. C-di-GMP levels in <i>M. tuberculosis</i> were manipulated by overexpressing a c-di-GMP synthesizing enzyme <i>ydeH</i> and a degrading enzyme <i>rv1357c</i>. We demonstrate that overexpression of <i>ydeH</i> lowers growth of the bacterium both <i>in vitro</i> and in <i>M. tuberculosis</i> grown in macrophages. This is in conformity with lowered expression of <i>mtrA</i> and selected genes of the <i>mtrA</i> regulon involved in cell wall turnover in the <i>ydeH</i>-overexpressing strain compared to the parent strain. We also demonstrate that overexpression of <i>ydeH</i> in <i>M. tuberculosis</i> hinders biofilm formation, whereas overexpression of <i>rv1357c</i> has the opposite effect. Neither of the two genes could rescue the biofilm defective phenotype of the MtrB knock out mutant (<i>ΔmtrB</i>), suggesting that c-di-GMP exerts its role on biofilm formation through MtrB. Finally, we show by fluorescence microscopy that the trafficking of <i>M. tuberculosis</i> overexpressing <i>ydeH</i> is significantly higher than that of the parent strain and that this is linked to reduced expression of the MtrB-dependent genes <i>esxG</i> and <i>esxH</i>, which play a role in subversion of lysosomal trafficking of <i>M. tuberculosis</i>. These results provide important new insight into the crosstalk between c-di-GMP and MtrB in <i>M. tuberculosis</i>.